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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2689-2697
By
From the Division of Hematology-Oncology, Department of Medicine, The
New York Hospital-Cornell Medical Center, New York, NY; and the
Division of Rheumatology, The Hospital for Special Surgery, New York,
NY.
Chronic lymphocytic leukemia (CLL) is characterized by a clonal
expansion of CD5+ B cells in the peripheral blood.
Associated immune aberrations include abnormal Th-cell
function and pathogenic autoantibodies. Under most circumstances, CLL B
cells do not proliferate in culture and express a limited repertoire of
surface antigens, including CD19, CD20, CD23, CD27, CD40, and CD70. In
this report, we demonstrate that freshly isolated B cells from a subset
of CLL cases constitutively express CD40 ligand (CD40L, CD154), a
member of the tumor necrosis factor family which is normally expressed
by activated CD4+ T cells and mediates T-cell-dependent
B-cell proliferation and antibody production. The degree of CD40L
expression varied considerably among the CLL cases examined. CD40L was
detected in purified CLL B cells by immunofluorescence flow cytometry,
by RT-PCR, and by immunoprecipitation. To demonstrate that CD40L in the
CLL B cells is functional, we used irradiated CLL cells to stimulate
IgG production by target, nonmalignant B cells in coculture. The CLL B
cells induced IgG production by normal B cells to a similar degree as did purified T cells in a process which was partially inhibited by
monoclonal antibody to CD40L. This is one of the first reports of CD40L
expression in a B-cell tumor. The data suggest that CD40L in the tumor
cells may be a factor in the generation of pathologic antibodies by
normal B cells in some patients with CLL.
CHRONIC LYMPHOCYTIC leukemia (CLL) is the
most frequent adult leukemia in the United States and is usually noted
due to an excess of well-differentiated B lymphocytes in the peripheral blood. The diagnosis is confirmed by fluorescence flow cytometry of the
lymphocytes, which characteristically express cell surface antigens
CD5, CD19, and CD23.1 Although the prognosis for early stage CLL is excellent, many patients with only a lymphocytosis require
treatment for and suffer complications from immune manifestations of
this disease.2-4 Autoimmune sequelae of CLL include both
Coomb's positive hemolytic anemia and thrombocytopenia.5,6
Independent of treatment, patients with CLL are vulnerable to
infectious pathogens due to impaired humoral immunity characterized by
hypogammaglobulinemia.7-10 A variety of T-cell derangements
have also been observed, which include a T-cell lymphocytosis, an
inverted ratio of CD4+ to CD8+ T
cells,11 deficient T-cell help,12,13 and
reduced capacity to secrete cytokines such as interleukin-2
(IL-2).14
CD40 ligand (CD40L; CD154, gp39, T-BAM)15 is
mainly expressed in activated, CD4+ T cells and, along with
CD28, is one of the main costimulatory signals by which specific,
antigen-reactive T cells offer help to B cells presenting that antigen
in the context of MHC-II.16-19 The receptor on the B cell
for CD40L is CD40, a member of the tumor necrosis factor (TNF) receptor
superfamily that is expressed in almost all B lymphocytes as well as in
macrophage and endothelial cells.20 Ligation of CD40 by its
ligand, CD40L, in the germinal center B cell results in inhibition of
apoptosis,21 proliferation, differentiation with Ig isotype
switching,22-24 and expression of activation antigens,
including CD2317,25 and Fas.26,27 As in most
B-cell tumors, CLL B cells express CD40,28-31 and several groups have observed that CLL B cells can be induced to differentiate into Ig-secreting cells.32-34
Normal T lymphocytes express CD40L for only a few hours after
activation. In T cells derived from patients with systemic lupus erythematosus (SLE), CD40L expression is increased and
prolonged.35,36 Several groups have described CD40L
expression in pathologic samples from certain T-cell malignancies and
also in human T-cell line cells.37,38 Whether human B cells
express CD40L is less established. Grammer et al39
demonstrated that human peripheral blood B cells could be induced to
express CD40L subsequent to stimulation with calcium ionophore and
phorbol ester, and that Epstein-Barr virus (EBV)-transformed
lymphoblastoid cell line cells (LCLs) express functional CD40L after
exposure to B-cell mitogens.39 Additional reports indicate
that CD40L is expressed in certain human B-cell tumors40
and in B cells from patients with SLE.36
In this work, we analyzed freshly purified CLL B cells and found that,
in a subset of CLL patient samples, there was significant CD40L
expression in the unstimulated malignant cells. CD40L in the CLL B
cells was functional in a costimulatory capacity, in that the CLL cells
induced antibody production by nonmalignant, human B cells. These
results provide evidence for a possible mechanism of paracrine tumor
stimulation in CLL and for excess autoantibody production due to
inappropriate B-cell "help."
Patient samples.
Approval for the research protocol was obtained from the internal
review boards of The New York Hospital and The Hospital for Special
Surgery, and verbal, informed consent was obtained before phlebotomy.
In each case, the diagnosis of CLL was established by the coexpression
of CD5 and CD19 in an expanded, clonal population of peripheral blood
lymphocytes. Mononuclear cells were isolated from 10 to 30 mL of fresh,
heparinized peripheral blood by Ficoll-Hypaque centrifugation. For most
of the experiments, T and B lymphocytes were separated by rosetting
with sheep red blood cells according to standard techniques, and the
degree of separation was checked by immunofluorescence flow
cytometry.
Cell culture.
Unless otherwise indicated, cells were analyzed immediately subsequent
to their purification. For some experiments, as indicated, cells were
cultured in C50 media (RPMI supplemented with penicillin-streptomycin with L-glutamine and 10% fetal calf serum [FCS]) only or with IL-2
at 40 U/mL (Hemagen Diagnostics, Waltham, MA) and a 1:60,000 dilution
of formalinized Staphylococcus Aureus Cowan 1 (SAC; Pansorbin; Calbiochem, San Diego, CA), recombinant IL-4 at 10 ng/mL (Genzyme Diagnostics, Cambridge, MA), monoclonal antibody (MoAb) to CD40 at 1 µg/mL (IgG Flow cytometry and immunofluorescence analysis.
Cells were washed in cold Hank's Buffered Saline Solution (HBSS) and
incubated with antibodies according to standard techniques. Cell
fluorescence was measured using a Becton Dickinson FACScan (Becton
Dickinson, San Jose, CA). Cells were assessed for forward and side
scatter, and events in the viable cell gate were analyzed for
fluorescence intensity using the CellQuest program (Becton Dickinson).
For acid washing of cells before analysis, the cells were exposed to
acidic PBS (pH 4.1) for 3 minutes at room temperature and then washed
twice with normal pH before exposure to MoAbs, as indicated, similar to
the technique reported by Grammer et al.39
MoAbs.
Sterile antibodies used in cell culture experiments included anti-CD40
(murine IgG1; Genzyme), anti-CD40L (murine
IgG1,, clone M-90; Genzyme), and anti-CD23 (EBVCS2; ATCC).
In the blocking coculture experiments, the dose of each antibody used
was 5 µg/mL. For single-color fluorescence flow cytometry, the
antibodies used were anti-CD3 (OKT3; ATCC), anti-V Reverse transcriptase-polymerase chain reaction (RT-PCR).
RNAs were isolated from approximately 1 × 107 cells
using the TRIzol reagent (Life Technologies/GIBCO BRL, Grand Island,
NY), according to the manufacturer's instructions, and
the yield in each case was measured by spectroscopy. For each sample,
cDNAs were prepared using approximately 1 µg of RNA, using a reverse transcriptase kit (GIBCO). For CD40L, the primers used were
5 Immunoprecipitation.
Freshly isolated purified CLL B cells were placed in culture under a
variety of circumstances. After 36 or 60 hours as indicated, the cells
were washed and cell surface proteins biotinylated using Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL).44 Lysates were
then subjected to immunoprecipitation overnight at 4°C using 3 µg
of antibody to CD40L (TRAP clone; PharMingen) or with 3 µg of a
control, isotype-matched antibody to IL-4 (Genzyme), in each case
conjugated to protein A Sepharose 4 Fast Flow beads (Pharmacia Biotech,
Piscataway, NJ). Precipitated materials were boiled for 4 minutes before sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride
(PVDF) membrane, exposed to horseradish peroxidase
(HRP)-conjugated streptavidin (Pierce) for 90 minutes, and
then imaged using a chemiluminescence substrate (Amersham,
Buckinghamshire, UK).
Ig measurement.
IgG and IgA secreted into the supernatants was measured by a standard
enzyme-linked immunoabsorbance assay (ELISA) protocol. In each
experiment, standard curves for the particular isotype were determined,
and the samples were diluted such that the absorbance readings fell in
the linear range of detection. For each circumstance and in each
experiment, antibody measurements were determined in triplicate.
Results were discounted if the standard deviation among the three
triplicate readings represented greater than 15% of the mean or if the
result for the diluted sample did not fall within the linear range of
the standard curve. IgG production, as shown in Fig 6, reflects the
results of single-case analyses, with error bars indicating the
standard error for measurements of each experimental circumstance.
CD40L expression is evident in some CLL B cells by immunofluorescence
flow cytometry.
CLL B cells from patients with untreated CLL were purified from
heparinized peripheral blood samples by Ficoll hypaque centrifugation and separated from the T lymphocytes by rosetting with sheep red blood
cells. Of the non-T cells obtained, greater than 96% of the cells in
each case expressed CD19. In the majority of cases, greater than 99%
of the purified cells were B cells. These cells were analyzed directly,
without stimulation, by immunofluorescence flow cytometry for B- and
T-cell antigens, including CD40L. In the first series
(Fig 1A), B cells from consecutive, unselected cases
were purified and examined using an MoAb to CD40L (clone M-90) or, as a
negative control, an isotype-matched control antibody to a T-cell
receptor (TCR)
CLL B-cell expression of CD40L is impacted by stimulation in vitro.
In cases that were positive, CD40L expression diminished rapidly over
time in culture with media only, such that the signal was less intense
48 hours after isolating the cells, became further diminished after 72 hours, and was undetectable thereafter (data not shown). However, some
stimuli did appear to augment CD40L in cases that had initially
undetectable levels. Figure 3 shows CD40L expression in
CD19+ cells from a single case that were treated for 48 hours after purification under a variety of experimental circumstances.
As shown in Fig 3B, the combination of IL-2 and SAC in culture was associated with increased CD40L expression in the CLL B cells. This
effect was observed in each of 5 cases for which we analyzed this
effect. In this particular sample, IL-4 did not result in augmented
CD40L expression (Fig 3C), but the effect of this cytokine has varied
among the 6 cases analyzed so far. Other B-cell stimulants also appear
to augment CD40L expression in the CLL cells. For example, signaling
induced by MoAb to CD40 (Fig 3D) and through ligation of the B-cell
surface antigen receptor (Fig 3E) both resulted in increased CD40L
expression at the cell surface.
CD40L message is readily detected in purified B cells from CLL
patients.
B cells were purified from 2 clinical CLL samples and placed in culture
for 16 hours before RNA extraction for analysis by RT-PCR. As shown in
Fig 4, CD40L message was readily detected in both of the
CLL samples exposed in vitro to media alone (lanes 1 and 5). In
addition, there was an increase in message in cells from the second
case after exposure to the combination of IL-2 and SAC (lane 6), to PMA
and ionomycin (lane 7), and to IL-4 (lanes 8), as compared with the
amplified signal for the housekeeping gene GAPDH. These results
correlated well with those obtained by immunofluorescence flow
cytometry.
CD40L can be detected in CLL samples by immunoprecipitation.
MoAb (TRAP clone) was used to precipitate CD40L from several samples.
For these experiments, lysates were prepared after biotinylation of
cell surface proteins. Material from 6 × 106 cells
was precipitated with either antibody to CD40L or with an
isotype-matched control antibody to IL-4 (Fig 5A). As
shown, we observed major bands at 39 and 32 kD corresponding to the
transmembrane forms of CD40L,46,47 which in this case were
most evident after exposure of the cells to IL-4 (lane 3). In addition,
a doublet was evident at 18 kD and another, single band at 15kD, as
have been reported48 and attributed to soluble
CD40L.49 The CD40L bands were not seen after
immunoprecipitation with a control antibody to human IL-4 (lanes 2 and
4). In a separate experiment, to control for the amount of protein in
each sample rather than cell number, CLL cells were stimulated for 60 hours with IL-2 and SAC and then examined for CD40L using 210 µg of
biotinylated protein in each sample (Fig 5B). As shown, there is a
prominent 39-kD band apparent in the stimulated cell lysate
precipitated with antibody to CD40L, but not with the control antibody.
CLL B cells do not proliferate spontaneously in vitro.
Other investigators have determined that ligation of CD40 results in
CLL B-cell activation31,49 and proliferation.50 In our laboratory, we performed thymidine incorporation assays to
examine CLL B-cell proliferation in culture under a variety of
circumstances. Given that the CD40/CD40L molecular pair is an important
modulator of CLL B cell growth, one might anticipate that the CD40,
CD40L-expressing tumor cells would proliferate spontaneously in
culture. However, as has been observed by other investigators, we did
not observe significant thymidine incorporation in any of the 6 cases
we studied without the addition of irradiated, CD40L+ T
cells (data not shown). These negative results suggest that other
factors besides CD40L, such as additional signals provided by accessory
molecules or T-cell-derived cytokines, are necessary for B-cell
proliferation in the context of CD40 ligation.
Capacity of CLL B cells to offer help via CD40L.
Because CD40L has a central role in T-cell-induced B-cell
differentiation and antibody formation, we investigated the capacity of
CLL B cells to trigger antibody production by normal, bystander B cells
in coculture. For these experiments, we used as targets B cells
purified from the peripheral blood of healthy volunteers. Effector
cells, used to stimulate antibody production by the target B cells,
included CLL B cells, autologous T cells obtained from the normal donor
in each experiment, and also T cells purified from the CLL patients.
These effector cells were irradiated and then placed in culture with
the target B cells as indicated in Fig 6. Supernatants
were removed and analyzed for IgG and IgA secretion by ELISA.
CD40L is primarily expressed in activated T cells and has not been
considered a regular feature of any B-cell tumor. We have observed that
freshly purified B cells from a significant proportion of CLL cases
express CD40L and can induce antibody production by target,
nonmalignant B cells in a process that is partially blocked by antibody
to CD40L. Although previous investigators have established that the
CD40/CD40L molecular pair is an important modulator of CLL B-cell
growth,28,49,50 we and other investigators have observed
that CLL B cells do not proliferate spontaneously in
vitro.49-52 Therefore, the extent to which CLL
B-cell-derived CD40L impacts tumor growth may depend on costimulation
via accessory molecules and the presence of T-lymphocyte-derived
cytokines, such as IL-4.50 Taken together, the data and the
literature suggest a model for CLL tumor growth and associated immune
aberrations due to tumor cells that, at least in some cases, express
both CD40 and CD40L (Fig 7).
Submitted October 2, 1997;
accepted January 23, 1998.
The authors thank the CLL patients and their physicians for providing
clinical samples. We thank Dr Radha K. Vakkalanka for expert guidance
in RT-PCR.
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Abstract
Introduction
Abstract
13, shaded histograms) or
with anti-CD40L (open histograms), followed by goat antimouse-FITC. In
the first case, the shaded histogram indicates CD3 expression. (B)
Peripheral blood mononuclear cells were washed briefly in neutral (pH
7.1) or acidic PBS (pH 4.1) to remove soluble CD40 from the
surface
1; Genzyme), an F(ab
)2
preparation of MoAb to human IgM at 0.5 µg/mL (Jackson Immunoresearch
Laboratories Inc, West Grove, PA), phorbol myristate acetate (PMA) at
10 ng/mL (Sigma) and ionomycin at 1.25 µg/mL (Calbiochem), or
pokeweed mitogen (PWM; Life Technologies, Gaithersburg, MD). For cDNA
preparation, Jurkat mutants that constitutively express CD40L (clone
D1.1) were used as a source of CD40L.
13 (murine
IgG
1), anti-CD19 (Immunotech, Westbrook, ME), anti-CD40 (Genzyme),
and anti-CD40L (clone M-90, murine IgG
) was 97% after culture with media alone and
98% after exposure to IL-4. In Fig